Design and Synthesis of Pyrrolo[2,1-a]Isoquinoline-Based Derivatives as New Cytotoxic Agents.

A new series of anti-cancer agents based on 1,2-diaryl-5,6-dihydropyrrolo[2,1-a]isoquinoline scaffold containing N,N-diethylamino-ethoxy, piperidinyl-ethoxy or morpholinyl-ethoxy group at the para position of the C-2 phenyl ring were synthesized and their cytotoxic activities were assessed against several human cancer cell lines including MCF-7 (ER positive breast cancer cell), MDA-MB231 (ER-negative breast cancer cell), T47D (Human ductal breast epithelial tumor cell line), A549 (adenocarcinomic human alveolar basal epithelial cells), and Hela (human cervix adenocarcinoma cells) using MTT assay. Based on results, compounds, 1-(4-(2-(piperidin-1-yl)ethoxy)phenyl)-5,6-dihydro-8,9-dimethoxy-2-phenylpyrrolo[2,1-a]isoquinoline (6a) and 2-(4-(5,6-dihydro-8,9-dimethoxy-2-phenylpyrrolo[2,1-a] isoquinolin-1-yl)phenoxy)-N,N-diethylethanamine (6c) were the most potent cytotoxic compounds and more toxic than the reference compound against T47D cell line, while all the compounds had satisfactory activity against HeLa cell line with mean IC50 values ranging from 1.93 to 33.84 µM.

Tamoxifen (TAM, 5) is well-known of a family of agents called selective estrogen receptor modulators (SERMs) which is widely used in the clinical management of primary and advanced breast cancer and as a preventive agent against recurrence after surgery since 30 years ago (12,13). The chemotherapeutic effect of tamoxifen is through prevention of binding of endogenous estrogen to the estrogen receptors within breast cancer cells and slowing the estrogen induced growth. Tamoxifen and many other SERMs could also induce cell death through apoptosis process. Regarding the anticancer properties of pyrrolo[2,1-a]isoquinoline compounds (Figure 1.) and anti breast cancer effects of tamoxifen, the aim of this study was to design new 1, 2-diaryl-5,6-dihydropyrrolo[2,1-a]isoquinoline derivatives possessing pharmacophoric basic side chain that might have expected anticancer activity. The cytotoxic activities of synthesized compounds were evaluated against several human cancer cell lines including MCF-7, MDA-MB231, T47D, A549 and Hela. For designing the new isoquinolines, the vicinal diaryl moiety and the basic side chain (N, N-dialkylaminoethoxyphenyl) was derived from the structure of tamoxifen while the total pyrrolo [2, 1-a] isoquinolines resembles the other cytotoxic isoquinoline compounds shown in Figure 1.

General
All chemicals and solvents used in this study were purchased from Merck AG and Aldrich except for tamoxifen obtained from (David Bull Labs, UK). Melting points were determined with a Thomas-Hoover capillary apparatus. Infrared spectra were acquired using a Perkin Elmer Model 1420 spectrometer. A Bruker FT-500 MHz instrument (Bruker Biosciences, USA) was used to acquire 1 H and 13 C NMR spectra with TMS as internal standard. Chloroform-D and DMSO-D 6 were used as solvents. Coupling constant (J) values are estimated in hertz (Hz) and spin multiples are given as s (singlet), d (double), t (triplet), q (quartet), m (multiplet), and br (broad). Low-resolution mass spectra were acquired with a MAT CH5/DF (Finnigan) mass spectrometer that was coupled online to a Data General DS 50 data system. The mass spectral measurements were performed on a 6410 Agilent LCMS triple quadruple mass spectrometer (LCMS) with an electrospray ionization (ESI) interface. Microanalyses, determined for C and H, were within ± 0.4% of theoretical values.

Chemistry
General procedure for the preparation of compounds 6a-d The DHIQ (Dihydroisoquinoline) analogs (compounds 6a-d) were synthesized as depicted in scheme1.

2-(4-acetoxyphenyl) acetic acid (2)
The mixture of (4-hydroxyphenyl) acetic acid dissolved in acetic anhydride using sulfuric acid as catalyst was refluxed for 15 min at 100 °C. After cooling, the mixture was set aside, poured into 200 g crushed ice and the resultant compound (the acetyl conjugate of 1) was obtained as a precipitate, which was filtered, washed with water and drained well. The crude acetyl conjugate (2) was then recrystallized from alcohol to give white crystalline powder. Yield: 48%; White solid; mp: 100 °C; IR (KBr):
The cells were cultured in RPMI1640 medium at 37 °C under 5% CO 2 supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 µg/mL streptomycin. Cell viability was assayed by using a MTT method which is based on the reduction of 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) dye to purple formazan crystals by mitochondrial succinate dehydrogenase enzyme in living cells. The cells were seeded into 96-well plates at a concentration of 10 4 cells/well and allowed to incubate for 24 h.
The cells were incubated with increasing concentrations of test compounds for 48 h. At the end of each treatment period, 10 μL of MTT (5 mg/mL in PBS) was added to each well and the microplate was incubated at 37 °C for 4 h. The medium with MTT was removed and 100 μL DMSO was added to each well to dissolve the insoluble formazan crystals. Plates were incubated for 20 min at 37 °C and the optical densities were read at 570 nm with a reference wavelength of 630 nm as background using a spectrophotometer plate reader (Infinite® M200, TECAN) (21). Tamoxifen was also used as positive control and DMSO as the solvent of the test compounds. Data are presented as the mean of triplicate number of living cells and their capacity to reduce samples. IC50 was calculated by calibration curve using Prism software.

Molecular modeling (docking) studies
Docking studies were performed using

Results and discussion
To determine the effect of synthesized compounds on the viability of breast cancer cells in vitro, ER-α-positive MCF7 and T-47D cells (containing medium to high levels of estrogen receptors), ER-α-negative MDA-MB-231 (without estrogen receptors) and two different cancer cell lines such as A549 (adenocarcinomic human alveolar basal epithelial cells) and Hela (human cervix adenocarcinoma cells) were used in MTT assay. To indicate the anti-proliferative activities of the designed compounds mediated through hormone-dependent or hormoneindependent mechanisms, the cells were treated with increasing concentrations of synthesized compounds (0-100 μM) and TAM (tamoxifen) (0-100 μM) as a reference drug.
The results of MTT assay are shown in Table 1. According to results, the synthesized compound 6b was the most cytotoxic compound against HeLa cell line while it was inactive against all types of breast cancer cell lines. The compounds 6a and 6c were cytotoxic against all cancer cell lines and showed the most potency against T47D cell line which contains high amounts of estrogen receptors in comparison with those of MCF7 and MDA-MB-231. This may be explained due to the ability of synthesized compounds to blockade estrogen receptors in breast cancer cell lines. In addition, our results showed that the presence of hydrophobic side chain such as piperidinyl or ethoxy increased cytotoxicity in comparison with compounds 6b and 6d containing polar morpholine group as polar side chain. However, our results showed that the synthesized compounds had also moderate to good cytotoxic activities on MDA-MB-231 cell line which indicated that other anticancer mechanisms may be involved in addition to blockade estrogen receptors in breast cancer cell lines.
Based on our MTT assay and structure similarity between designed 1, 2-diaryl-5, 6-dihydropyrrolo [2,1-a] isoquinoline compounds 6a-d and tamoxifen, it could be assumed that one of the mechanisms for cytotoxic activity of compounds 6a-d on breast cancer cell lines is mediated through estrogen receptors. Therefore, the orientation of 2-(4-(5, 6-dihydro-8, 9-dimethoxy-2phenylpyrrolo[2, 1-a] isoquinolin-1-yl) phenoxy)-N, N-diethylethanamine 6c as the most potent compound against T47D, in the estrogen receptor α (ERα) active site was examined by a docking experiment (Figure 2). This molecular modeling study showed that compound 6c was well bound into the active site of ERα so that the N atom of the tertiary amino group of the basic side chain (diethylaminophenoxy) is in the vicinity of the oxygen of carboxylate group of Asp 351 (distance = 4.01 Å) and is capable of binding to this amino acid. The methoxy group on phenyl ring is also in the vicinity of Glu 353 so that the O atom of methoxy group is in a 2.6 Å distance from the OH of carboxylic group in Glu 353 (24). In addition, molecular modeling studies (Figure 3.) showed the good superimposition of compound 6c with lasofoxifen as a crystallography compound in the  estrogen receptor active site. These data together with biological results are in agreement that one of the mechanisms of cytotoxic activity of compounds 6a and 6c on breast cancer cell lines might be mediated through acting on estrogen receptors.

Conclusion
This study indicates that all synthesized compounds showed significant cytotoxicity against HeLa cell line while compounds 6a and 6c demonstrated cytotoxicity against all types of breast cancer lines. In addition, modifications on the basic side chain of 1, 2-diaryl-5, 6-dihydropyrrolo [2,1-a] isoquinoline scaffold had a significant influence on the cell cytotoxicity.
work as part of Ph.D thesis of Samaneh kakhki.

Authors› Statement
The authors declare no conflict of interest. (13)